The T cell growth factor IL-2 is the major cytokine produced during the primary response of T helper (Th) cells. Upon differentiation into one of the two types of Th effector cells, Th1and Th2, IL-2 production declines and is replaced by production of Th1-like (IFNγ) or Th2-like (IL-4) cytokines. IL-2 acts through its receptor (IL-2R) to activate signaling molecules involved in cell proliferation; defects in either the ligand or the receptor result in autoimmunity (Schimpl, A., I., et al. 2002. Cytokine Growth Factor Rev 13:369-378). Although IL-2 has been previously characterized as a Th1-like cytokine, increasing evidence indicates that IL-2 and its downstream signaling molecule Stat5 are also vital for the induction of anti-inflammatory Th2 cytokines during a primary response (Zhu, J., J., et al. 2003. Immunity 19:739-748).
IL-2 expression is tightly controlled at the transcriptional level although posttranscriptional control through coding sequences also occurs (Ragheb, J. A., et al. 1999. J Immunol 163:120-129). Extensive analysis of the IL-2 gene has established a minimal promoter region extending -300 bp relative to the transcription start site, known to be sufficient for IL-2 induction upon T cell activation in vitro (Durand, D., et al. 1988. Mol. Cell. Biol. 8:1715-1724; Siebenlist, U., et al. 1986. Mol. Cell. Biol. 6:3042-3049) (and reviewed in Jain, J., C et al. 1995. Curr Opin Immunol 7:333-342; Serfling, E., et al. 1995. Biochim Biophys Acta 1263:181-200; Powell, J. D., et al. 1998. Immunol Rev 165:287-300; Novak, T. J., P et al. 1990. Nucleic Acids Res 18:4523-4533). Multiple cis regulatory elements within this region have been identified that bind antigen-inducible factors such as NFATs, OCT-1, AP-1, HMG I(Y) and NF-κB family members p65 and c-Rel. These factors have been shown to transactivate an IL-2 promoter in transient reporter assays (reviewed in Jain, J., C et al. 1995. Curr Opin Immunol 7:333-342; Serfling, E., et al. 1995. Biochim Biophys Acta 1263:181-200; Powell, J. D., et al. 1998. Immunol Rev 165:287-300; Novak, T. J., P et al. 1990. Nucleic Acids Res 18:4523-4533) and some of them are required for IL-2 expression in vivo (Peng, S. L., et al. 2001. Immunity 14:13-20; Kontgen, F., et al. 1995. Genes Dev. 9:1965-1977; Liou, H. C., et al. 1999. Int. Immunol. 11:361-371). NF-κB family members regulate the transcription of the IL-2 gene (Jain, J., C et al. 1995. Curr Opin Immunol 7:333-342; Serfling, E., et al. 1995. Biochim Biophys Acta 1263:181-200; Powell, J. D., et al. 1998. Immunol Rev 165:287-300; Novak, T. J., P et al. 1990. Nucleic Acids Res 18:4523-4533). While p50/p50 homodimers are present in large amounts in unstimulated cells, they are inhibitory and are replaced by p50/p65 or p50/c-rel heterodimers upon T cell activation. c-Rel nucleates chromatin remodeling across the IL-2 promoter (Grundstrom, S., et al. 2004. J Biol Chem 279:8460-8468; Lai, J. H., et al. 1995. Mol Cell Biol 15:4260-4271; Neumann, M., et al. 1995. Embo J 14:1991-2004; Ghosh, P., et al. 1993. Proc Natl Acad Sci USA 90:1696-1700; Parra, E., et al. 1998. J Immunol 160:5374-5381; Herndon, T. M., et al. 2002. Clin Immunol 103:145-153; Rao, S., et al. 2003. J Immunol 170:3724-3731; Kahn-Perles, B., et al. 1997. J Biol Chem 272:21774-21783). Interestingly, increased amounts of the NF-κB p65 (RelA) factor in the nucleus of Th1 than Th2 cells has been reported, consistent with the preferential secretion of IL-2 by Th1 cells (Lederer, J. A., et al. 1994. J Immunol. 152:77-86; Dorado, B., et al. 1998. Eur J Immunol 28:2234-2244).
Lines of transgenic mice revealed a requirement for additional IL-2 upstream sequence to achieve expression in vivo that faithfully mirrors endogenous IL-2 expression (Yui, M. A., et al. 2001. J Immunol 166:1730-1739). The contribution of regions beyond the minimal promoter is also evident from studies that show that selective demethylation of a 600 bp region of an IL-2 enhancer occurs rapidly upon T cell activation (Bruniquel, D., and R. H. Schwartz. 2003. Nat Immunol 4:235-240). The function of individual factors that bind IL-2 promoter DNA and the initiation of chromatin remodeling of the IL-2 gene in response to T cell activation has been the subject of several reports (Ward, S. B., et al. 1998. Nucleic Acids Res 26:2923-2934; Rothenberg, E. V., and S. B. Ward. 1996. Proc. Natl. Acad. Sci. USA 93:9358-9365; Attema, J. L., et al. 2002. J Immunol 169:2466-2476; Chen, X., et al. 2005. Mol Cell Biol 25:3209-3219; Rao, S., et al. 2001. J Immunol 167:4494-4503). The NF-κB subunit c-Rel is required for chromatin remodeling across the proximal promoter and c-Rel binds with HMG I(Y) to the CD28 response element (Rao, S., et al. 2003. J Immunol 170:3724-3731; Himes, S. R., et al. 1996. Immunity 5:479-489). Mice lacking c-Rel exhibit impaired IL-2 expression, and treatment with the c-Rel inhibitor pentoxifylline reduces IL-2 mRNA levels (Kontgen, F., et al. 1995. Genes Dev. 9:1965-1977; Liou, H. C., et al. 1999. Int. Immunol. 11:361-371; Wang, W. W., et al. 1997. Immunity6:165-174).
Negative regulation of IL-2 gene transcription is also an important mechanism for controlling its expression. During primary Th1 cell differentiation, IL-2 is rapidly induced and peaks between day 2 and day 3 post TCR stimulation, then gradually decreases. Homodimers of the NF-κB member p50 are thought to repress IL-2 gene transcription in resting Th cells (Grundstrom, S., et al. 2004. J Biol Chem 279:8460-8468; Sundstedt, A., et al. 1996. Proc Natl Acad Sci USA 93:979-984) and expression of a dominant negative CREB transgene resulted in impaired IL-2 production in vivo (Barton, K., et al. 1996. Nature 379:81-85). The CREM transcriptional repressor is activated by CaMKIV to bind to a CRE at position-180 to suppress IL-2 production in patients with SLE (Juang, Y. T., et al. 2005. J Clin Invest 115:996-1005; Tenbrock, K., et al. 2002. J Immunol 169:4147-4152), and CREM is also involved in establishing the anergic state (Powell, J. D., et al. 1999. J Immunol 163:6631-6639). A zinc finger protein named ZEB is thought to be a transcriptional repressor of the IL-2 gene, but its function in primary Th cells has not been established (Yasui, D. H., et al. 1998. J. Immunol. 160:4433-4440). The antiproliferative factor Tob represses IL-2 through enhancing Smad binding to the -105 negative regulatory element of the IL-2 promoter (Tzachanis, D., et al. 2001. Nat Immunol 2:1174-1182).
The T-box transcription factor, T-bet, has three separable functions: 1) it is required for Th1 development from the Thp, 2) it represses Thp differentiation along the Th2 pathway by inhibiting GATA-3 activity through the physical interaction of tyrosine phosphorylated T-bet and GATA-3, and 3) it represses IL-2 gene activation (Szabo, S. J., et al. 2000. Cell 100:655-669; Szabo, S. J., et al. 2002. Science 295:338-342; Szabo, S. J., et al. 2003. Ann. Rev. Immunol. 21:713-758; Hwang, E. S., S et al. 2005. Science 307:430-433). Consequently, T-bet-/- mice exhibit impaired Th1 cell development, increased Th2 cytokine production, and interestingly, increased IL-2 production in both CD4 and CD8 cells (Szabo, S. J., et al. 2002. Science 295:338-342; Sullivan, B. M., et al. 2003. Proc Natl Acad Sci USA 100:15818-15823; Juedes, A., et al. 2004. J. Exp. Med. 199:1153-1162). Indeed, T-bet was originally isolated in a yeast one hybrid screen that utilized the 400 bp IL-2 promoter as substrate and was subsequently shown to repress IL-2 promoter activation (Szabo, S. J., et al. 2000. Cell 100:655-669). Further, overexpression of T-bet in T-bet-/- Th cells repressed IL-2 production (Szabo, S. J., et al. 2000. Cell 100:655-669; Hwang, E. S., S et al. 2005. Science 307:430-433). While the first two functions of T-bet are understood at a molecular level, the mechanism by which T-bet controls production of IL-2 has not been apparent. The identification of the mechanism by which T-bet controls production of IL-2 would be of great benefit.